Structural Characteristics And Catalytic Functions Of Salix Babylonica Glutathione Transferases

Phytoremediation plants contribution to environmental pollutant remediation via various channels including absorption,accumulation,fixation,and decomposition.The growth and physiological activity of plants would be inhibited when the tolerations are exceeded.Plant-specific tau glutathione transferases(GST)are basically involved in catalyzing glutathione(GSH)-dependent conjugation reactions with pesticides and herbicides,which play an important role in the detoxification of pollutants.Salix babylonica has been the great detoxification species.There is no exploration of the functional characteristics of GST,and the lack of systematic biochemical and structural researches of tau GST.In general,this study not only provided the systematically studied for Sb GST but also revealed the catalytic mechanism and structural characteristics of the toxic compounds.The main results are as follows:1.In this study,22 full-length GST genes were cloned from Salix babylonica and divided into three classes based on the conserved domain analysis,phylogenetic tree,and gene structure: tau,phi and DHAR.The tau GST contained the highest number of members among these Salix GST.In addition,this study confirmed that the sequences similarities between the N-terminal domain sequences were higher than those of the C-terminal domain sequences.The Salix tau and phi GST had a broad substrate spectrum and differentiated substrate activities.The identical gene cluster members have different substrate spectrum.The substrate activities of different Salix GST also had significant differences for the identical substrate.2.Among the 14 Salix tau GST,Sb GSTU6 and Sb GSTU7 showed the highest and lowest enzymatic activities,respectively.Evolutionary conservation analysis revealed that that the Thr53 of Sb GSTU7 was a G-site residue,while this residue was Ile/Val in all the other Salix tau GST.Site-directed mutagenesis experiments showed that the enzymatic activity of Sb GSTU7(T53I)and Sb GSTU7(T53V)mutants were significantly higher than wild type.These results indicated that the Ile/Val→Thr substitution could result in the low enzymatic activities of Sb GSTU7.3.The catalytic mechanisms of Sb GSTU6 and Sb GSTU7 were fitted well to the simplest rapid equilibrium,random sequential Bi Bi model.Moreover,Sb GSTU6 and Sb GSTU7 possessed different affinities towards GSH at fixed CDNB or NBD-Cl concentrations.Hence,the binding of hydrophobic substrate could lead to the change in GSH binding capability,which further proves that the catalytic mechanism of Sb GSTU6 and Sb GSTU7 was a random mechanism rather than a sequential mechanism.4.The steady-state kinetic analyses found that the mutation into Thr was not conductive to GSH binding.This process also resulted in a significant decrease in the catalytic efficiency against various substrates,making the formation of enzyme substrate(ES)complexes more difficult.The ITC analysis showed lower dissociation constants(Kd)in Sb GSTU6 and Sb GSTU7(T53I),which further confirmed that the Ile residue could enhance protein–ligand affinity.Therefore,the presence or removal of a hydroxy group at this position affected GSH binding,probably due to the molecular rearrangement of the G-site.5.The results of PROPKA calculation suggested that the p Ka value of Glu67 in Sb GSTU6 was significantly higher than that of its mutant.An identical phenomenon was observed in Glu65 of Sb GSTU7(T53I).The substitution to Ile also caused the optimum p H value to shift towards a more alkaline environment.In addition,our results showed that Glu67(Glu65 of Sb GSTU7)possessed the identical p Ka value with GSH entry.In the presence of Thr residues,the p Ka value was relatively low,and the G-site needed more dissociation to promote the increase in environmental p H,which was unfavourable to the catalysis reaction.The reduced reactivity may also be due to the fact that the amino group of the γ-L-Glu could not interact with the cysteinyl thiol group,thus showing a lower p Ka value.6.In this study,the viscosity kinetic experiments showed that the rate-limiting step of the GST systems with conserved Ile was more significantly limited by the conformational change.These results implied that the most important factors affecting the GST enzymatic activity may have originated from structural stability and flexibility.This phenomenon was also demonstrated by our crystallography study and MD simulations.The non-synonymous substitution resulted in the rearrangement of the GST active cavity,reducing the affinity and catalytic efficiency.The thermal stability and RMSD results showed that the presence of Ile enhanced the conformation flexibility of the Sb GSTU proteins and was advantageous to GSH binding.In conclusion,our systematic and exhaustive evidence revealed the evolution and functional characteristics of Salix babylonica GST,and the critical role of the Ile residue at the G-site on the structural and catalytic features.This study not only provides the structural basis for GST engineering in agricultural biotechnology but is also beneficial for the design of antidotes against environmental pollutants.